The present invention relates to the identification and utilization of promoters for expression of nucleic acid sequences in thermophiles.
The high-temperature operating conditions of certain industrial processes in the areas of pharmaceutical synthesis, biodegradation of complex agricultural and industrial waste compounds, and food processing dictate the use of thermostable ENZYMES that can function at high temperatures. A significant advantage thermostable enzymes provide are cost savings resulting from longer storage stability and the higher activity at high temperature.
Thermostable enzymes have traditionally been used for saccharification in food processing and proteolysis in detergent industry (Williams, R. A. D. Biotechnological applications of the genus Thermus. Thermophiles: Science and Technology, Reykjavik, Iceland, 1992). Glucosidases are utilized extensively throughout the starch processing industry. Thermostable carbohydrases are directly involved in the manufacture of all starch-derived products. Isomersases are involved in production of high-fructose corn syrup. Two other important industrial carbohydrases are the pectolytic enzymes and lactase (Burgess, K. and M. Shaw. In Industrial enzymology, Ed. by T. Godfrey and Reichelt, J., N.Y. p. p. 260. 1983; Bombouts, F. M. and W. Pilnik. In Microbial enzymes and bioconversions. Ed. by A. H. Rose, N.Y. p. 269. 1980). A recent development in the industrial enzyme area is the use of cellulase for the production of glucose from cellulose (Mandels, M. In Annual reports on fermentation processes. Ed. by G. T. Tsao, N.Y. p. 35. 1982). Proteolytic enzyme, constituting a significant segment of the total industrial enzyme market are utilized in the detergent industry (Godfrey, T. and J. Reichelt. (1983) Industrial enzymology.).
One of the most promising new applications of thermostable enzymes is in the manufacture of specialty chemicals and pharmaceutical intermediates. Enzymes (or biocatalysts) are now being viewed as clearly superior in cases where stereospecific synthetic reactions are involved, such as synthesis of chiral compounds as pharmaceutical intermediates. Enzymes can carry out the reaction more specifically and under conditions which are safer for the environment. Thermostable enzymes have advantages since they are generally more stable in organic solvents, can carry out reactions at high temperatures where substrate and product solubility is higher, and can be recycled and used for longer periods of time because of their inherent stability.
A relatively new application of thermostable enzymes is PCR-based diagnostics. Thermostable polymerases have been extremely useful in the detection and molecular characterization of agents causing cancer, AIDS, and numerous other infectious diseases. Thermostable DNA-polymerase can already compete in market value with enzymes having traditional applications. Thermostable DNA replication proteins have important applications in molecular biology research.
A significant limitation to the widespread use of thermostable enzymes in such applications is the difficulty in expressing the proteins in a host bacteria. Thermophilic bacteria belong to a wide range of very different taxonomic groups (Kristjansson, J. K. and K. O. Stetter. Thermophilic bacteria. In Thermophilic bacteria. Ed. by J. K. Kristiansson, CRC Press, Inc., Boca Raton. p. 1-18. 1992). One of the best studied is the gram negative genus Thermus. Species belonging to this genus are easy to handle, growing aerobically in a broad temperature range of 45 to 85xc2x0 C. and not requiring pressurized incubation devices (Williams, R. A. D. Biotechnological applications of the genus Thermus. Thermophiles: Science and Technology, Reykjavik. Iceland, 1992). These strains grow to high densities in simple and inexpensive liquid media and form colonies on solid agar. With a doubling time less than 2 hours, the microorganisms of the genus Thermus are suitable organisms for various industrial applications.
Despite the widespread interest in Thermus us cultures for a variety of applications, there is a need for systems to control gene expression in Thermus. Currently the expression of heterologous genes in thermophilic hosts is difficult and inconvenient. Expression vectors for thermophiles do not provide a choice of promoters or ribosome binding sites, nor do they provide convenient ways to regulate expression. The reagents and methodologies provided herein allow for the production of commercially important enzymes including those from hyper- and extreme-thermophiles that can be difficult to produce using other systems. Also provided are reagents and methodologies for the construction of high-temperature fermentation strains which have been metabolically engineered with exogenous DNA for use in bioprocess applications such as production of complex molecules and pharmaceutical intermediates. Additionally, the systems provided herein are useful for thermostabilization of mesophilic proteins by genetic selection in a thermophile, which can also lead to altered enzymatic activity and a better understanding of the biochemical determinants for thermostability.